This project conducts clinical trials of live intranasal pediatric vaccine candidates that we have made for human respiratory syncytial virus (RSV), human parainfluenza virus serotypes 1, 2, and 3 (HPIV1, 2, and 3), and human metapneumovirus (HMPV). We presently are focusing on RSV. We are conducting Phase 1 pediatric clinical trials of multiple live-attenuated RSV strains of our design. The goal is to identify one or two lead candidates suitable for further development as an intranasal pediatric RSV vaccine. This is being done under a Cooperative Research and Development Award (CRADA) with Sanofi Pasteur, Inc. One lineage of live-attenuated RSV vaccine candidates that has been under development involves attenuation mainly by point mutations, and is exemplified by the virus rA2cp248/404/1030delSH. Previously, this virus appeared to be appropriately attenuated and moderately immunogenic in 6-24 month old seronegative children and 1-2 month old RSV-naive young infants, and was our previous lead candidate. However, this virus exhibited genetic instability that primarily involved two major attenuating temperature-sensitivity (ts) missense mutations in the L protein called 248 and 1030 (which involve L codons 831 and 1321, respectively). We previously developed new versions of these two mutations that were modified for increased genetic stability (called 248s and 1030s). We then incorporated these two stabilized mutations together with the other mutations of rA2cp248/404/1030delSH into a new virus called RSV cps2. This virus has now been evaluated in a Phase 1 pediatric clinical trial in seronegative children 6-24 months of age (NCT01852266 and NCT01968083). This virus was highly attenuated. Sequence analysis of shed vaccine virus from vaccinees did not detect any de-attenuation at the stabilized attenuating mutations 248s or 1030s, confirming that substantial stabilization of these mutations indeed had been achieved. Unfortunately, the cps2 virus proved to be somewhat more attenuated than the previously-evaluated rA2cp248/404/1030delSH virus. We believe the greater attenuation of cps2 is because this virus was constructed using a backbone that had a number of other point mutations compared to rA2cp248/404/1030delSH. At the time cps2 was made, these additional mutations were thought to be incidental, but more recently we have been finding that seemingly-incidental differences in ostensibly-wild-type backbones can be associated with increased attenuation, and this likely is the case with cps2. Therefore, we believe that the incorporation of the 248s and 1030s mutations into the exact rA2cp248/404/1030delSH backbone would create a more stable version that retains the desirable attenuation phenotype of rA2cp248/404/1030delSH. This virus will be made, but with regard to clinical trials we will focus on other vaccine virus lineages (see below) that may have more advantageous properties. The stabilized 248 and 1030 mutations, as well as a stabilized codon-deletion mutation (del1313/I1314L) that we also developed, also are available for use in other backbones; for example, the 1030s and del1313/I1314L mutations have been combined with the NS2 and M2-2 gene-deletions in other vaccine candidates (see below, and results not shown). A second lineage of RSV vaccine candidates involves deletion of the ORF encoding the small (90 amino acids) viral M2-2 protein (delM2-2 lineage). The M2-2 protein plays a role in regulating RSV RNA synthesis, and its deletion results in down-regulated viral RNA replication (causing viral attenuation) and up-regulated viral gene transcription and antigen synthesis. Increased antigen expression per genome raises the possibility of increased immunogenicity per infectious particle. We have been evaluating several versions of delM2-2 viruses in Phase 1 clinical trials in RSV-seronegative children 6-24 months of age. A prototype virus called RSV MEDI/delM2-2 was evaluated in seronegative infants 6-24 months of age (NCT01459198). This virus was very highly attenuated for replication, and possibly was more attenuated than necessary. Nonetheless, compared to the rA2cp248/404/1030delSH virus described above, the MEDI/delM2-2 virus induced significantly higher titers of serum RSV-neutralizing antibodies. Thus, it indeed appeared to have increased immunogenicity per infectious particle. Surveillance during the subsequent RSV season provided presumptive evidence of protection against wild-type RSV infection, as well as strong anamnestic RSV-specific antibody responses. A second delM2-2-based candidate, called RSV LID/delM2-2, was evaluated in similar Phase 1 studies (NCT02040831 and NCT02237209). Unexpectedly, the LID/delM2-2 virus was substantially less restricted than MEDI/delM2-2, and may be insufficiently attenuated. The difference in attenuation between these two viruses appears to involve a number of sequence differences in the two viral backbones. The LID/delM2-2 virus was more immunogenic than MEDI/delM2-2, presumably due to its increased replication. Because MEDI/delM2-2 may be more attenuated than needed, while LID/delM2-2 may be under-attenuated, we sought to identify a delM2-2 virus that was intermediate between the two. Thus, several other delM2-2 viruses presently are under evaluation in small Phase 1 trials, namely RSV D46/cp/delM2-2 (NCT02601612), RSV LID/delM2-2/1030s (NCT02794870 and NCT0252339), RSV LID/cp/delM2-2 (NCT02890381 and NCT02948127), RSV D46/NS2/N/delM2-2 (NCT03099291 and NCT03102034), and RSV 276 (NCT03227029 and NCT03422237). These studies are in progress. A third lineage of RSV vaccine candidates contains deletion of the NS2 gene (delNS2 lineage), whose encoded protein antagonizes host responses to viral infection, notably the type I interferon (IFN) and apoptosis responses. In one candidate, the delNS2 mutation was combined with the del1313/I1314L mutation, creating the virus RSV delNS2/del1313/I1314L. This virus is being evaluated in Phase 1 pediatric clinical trials, in one case on its own (NCT01893554), and in a second case in a head-to-head comparison with the delM2-2 virus called RSV 276 noted above (NCT03227029 and NCT03422237). In a second candidate, the delNS2 mutation was combined with the stabilized 1030s mutation noted above (which consists of Y1321K and S1313(TCA) in the L ORF). This created the virus RSV delNS2/1030s. This virus also is being evaluated in a Phase 1 pediatric clinical study (NCT03387137). The 1030s mutation is somewhat less attenuating than the del1313/I1314L mutation, and thus these two viruses should exhibit a range of attenuation phenotypes. A fourth lineage of RSV vaccine candidates contains deletion of the NS1 gene (delNS1 lineage) that, like NS2, encodes a protein that antagonizes host interferon and apoptosis responses, but does so more efficiently than NS2 and thus might confer a phenotype that is more attenuated and immunogenic. Two viruses were made that each contain the delNS1 deletion as the sole attenuating element, but in one virus the F and G genes have been moved to the first and second genome positions in order to increase their expression (RSV 6120/delNS1 and 6120/F1G2/delNS1, respectively). These viruses presently are being compared head-to-head in a Phase 1 pediatric clinical trial (NCT03596801). In collaboration with the Medical Virology Section, cDNA-derived RSV strain A2 presently is being evaluated in an in-patient setting for infectivity, replication, pathogenesis, and immunogenicity in healthy adult volunteers in a dose-escalation study (NCT02484417). This will provide an infection model that can be used to evaluate anti-RSV drug candidates and adult RSV vaccine candidates, and to study viral pathogenesis and the host response.